Recommended problems from chapter 18: 3,5,7,8,9,10,11,12,13,14,16,17,21,22

Glycolysis is the sequence of reactions that are used to break down . Glycolytic reactions are conserved through most organisms, so the sequence of reactions (including substrates, products, and ) is almost the same in all organisms. Clearly, this is a successful approach to .

Glycolysis overview

Steps 1 and 3 involve energy investment in the form of ATP .

Step 6 generates 2 NADH molecules

Steps 7 and 10 together generate 4 ATP molecules.

1 Reaction 1. ATP + Glucose → Glucose-6- + ADP + H+ This step depends on hexokinase and . A is an that transfers a phosphoryl group to a . Hexokinase can transfer a phosphoryl group to many 6-carbon , such as D- and D-. catalyzes the same reaction in the , and this enzyme is primary involved in maintaining blood sugar levels.

The first step - hexokinase ATP + Glucose → Glucose-6-phosphate + ADP + H+

Magnesium are required for hexokinase activity. Magnesium “shields” or “engages” the negative charges of the atoms and makes the γ- more susceptible to nucleophilic attack by the C6 hydroxyl oxygen. Uncomplexed ATP is an inhibitor of hexokinase activity. Would you expect uncomplexed ATP to be a competitive or a non-competitive inhibitor of hexokinase?

2 Water is excluded from the of hexokinase in the presence of glucose

Uncomplexed enzyme + glucose complexed enzyme:glucose

Why is it favorable to exclude water from the active site during phosphoryl transfer?

Reaction 2. Phosphoglucose

Both G6P and F6P exist predominantly in a closed ring structure, so the reaction requires ring opening, isomerization, then ring closure.

3 Proposed mechanism of phosphoglucose isomerase

Reaction 3 -

The uncoupled reaction is given by:

Fructose-6-P + Pi → Fructose-1,6-bisphosphate ∆G°’ = +16.3 kJ/mol

If ATP hydrolysis is coupled to the reaction, the reaction becomes

Fructose-6-P + ATP → Fructose-1,6-bisphosphate + ADP ∆G°’ = -14.2 kJ/mol

This reaction “commits” the reaction to go to the right under standard conditions.

4 Reaction 3. Regulation of PFK by ATP

Is ATP an inhibitor or activator of PFK? Does this make sense in terms of the final outcome of the pathway?

Reaction 3. Regulation of PFK by AMP AMP is a positive allosteric effector (an activator) of PFK. The cellular concentrations of AMP, ADP and ATP are regulated by the enzyme : ADP + ADP ↔ AMP + ATP Keq = 0.44

Typical ADP levels are 9% of ATP, and AMP levels are less than 1% of ATP. Because of this equilibrium, the concentration of AMP can rise very fast as a result of ATP hydrolysis. For example, by how much will the concentration of AMP rise if the concentration of

ATP falls by 10% as a consequence of hydrolysis into ADP + Pi? Assume that the typical concentration of ATP is 1.5 mM.

This is an example of an exceptionally elegant feedback regulatory circuitry. As [ATP] is decreased, [AMP] is increased rapidly due to the equilibrium above. This information is “read” by the glycolytic pathway as a signal to increase production of ATP.

5 Reaction 3. Regulation of PFK by citrate

Citric acid is a negative allosteric effector of PFK. This achieves a coupling of the cycle and glycolysis. Glycolysis “feeds” the by the production of pyruvate and acetyl CoA. Because the main function of the citric acid cycle is the production of ATP, when this pathway is saturated glycolysis is slowed such that glucose is not committed into glycolysis as fast.

Reaction 4. Aldolase

Cleavage of F-1,6-BP into DHAP and GAP. Note the carbonyl at C2.

6 Uncatalyzed aldol cleavage

The enolate transition state is stabilized by the multiple resonances. This is due to the ability of the carbonyl oxygen to withdraw . The carbonyl at C2 of FBP is a consequences of reaction 2, the isomerization of G6P to F6P. This is beneficial because the aldolase reaction with F6P yields two transition state compounds that can be interconverted and thereby stabilize the transition state.

What catalytic strategy(ies) would you expect to be used by an enzyme to facilitate the rate of this reaction?

Reaction 4. Mechanism of class I aldolase - and cells

1. Substrate binding.

7 Mechanism of class I aldolase - animal and plant cells 2. Chain opening and schiff-base formation between the enzyme and the open chain.

•The enzyme + substrate complex is inactivated by the presence of NaBH4 due to the transfer of a hydride to the imine carbon. The resulting ES complex is stable and does not go through subsequent steps (what type of inhibitor is NaBH4?).

•Incubation of a 14C-labeled substrate with the enzyme + NaBH4 followed by complete of the enzyme to yield individual amino acids can be used to identify a modified lysine residue.

•Incubation of the enzyme alone with NaBH4 followed by removal of NaBH4 results in no inhibition.

3. intermediate formation within class I aldolase Step 3 of this reaction results in aldol cleavage, release of GAP, and Enamine-enzyme covalent complex formation. The iminium ion is a better electron withdrawing group than the carbonyl oxygen of the precursor. The enamine intermediate is more stable than the enolate intermediate of the uncatalyzed reaction, and this results in a substantial rate enhancement in the enzyme-catalyzed reaction.

8 4. Protonation of the enamine intermediate within class I aldolase results in iminium ion (protonated Schiff base) formation

5. Hydrolysis of the iminium ion and release of DHAP

9 Class II aldolases in fungi, algae, and

The enolate intermediate is stabilized by a zinc or (II) ion within the enzyme.

GAP (an ) DHAP (a ) Reaction 5. phosphate H isomerase (TIM) H O C H C OH The two 3-carbon products of are H C OH C O interconverted by TIM. - - The enediol intermediate pathway is CH2OPO3 CH2OPO3 supported by the binding of the transition state analogs phosphoglyco- hydroxamate and 2-phosphoglycolate to the enzyme. The transition state analogs bind the H OH enzyme with 100 to 155 fold increased C affinity, in comparison to the an enediol intermediate physiological substrates. This allows a C OH quantitative assessment of the transition CH OPO - state stabilization achieved by the 2 3 enzyme. OH

- N - O O O


- - CH2OPO3 CH2OPO3 Phosphoglyco- 2-Phosphoglycolate hydroxamate

10 Summary of the first stage of glycolysis

The second stage of glycolysis - generation of additional high energy compounds

Reaction 6. Oxidation and of GAP By -3- phosphate . The favorable oxidation reaction contributes free energy in order to drive the generation of the high-energy acyl phosphate and reduction of NAD+ into NADH. Overall, however, the reaction is slightly unfavorable under standard conditions.

11 Mechanistic studies of GAPDH

1. GAPDH is inhibited by alkylation with stoichiometric amounts of iodoacetate, and a carboxymethylcysteine is present among the hydrolysis products of the modified enzyme. The enzyme must contain an essential residue.

2. GAPDH transfers 3H from the C1 of GAP to NAD+.

Mechanistic studies of GAPDH

32 3. GAPDH can exchange P from Pi in solution to the analog acetyl phosphate.

What substrate binding mechanism is supported by these results?

12 Catalytic mechanism of GAPDH, proposed by D. Trentham 1. Substrate binding.

Catalytic mechanism of GAPDH, proposed by D. Trentham 2. The essential sulfhydryl group acts as a , attacks the aldehyde, and a thiohemiacetal is formed.

13 Catalytic mechanism of GAPDH, proposed by D. Trentham 3. An acyl thioester is formed by oxidation of the thiohemiacetal. H+ is directly transferred to NAD+. This is a covalent high-energy intermediate that has been isolated.

Catalytic mechanism of GAPDH, proposed by D. Trentham

4. NADH dissociates and NAD+ binds.

5. 1,3-bisphosphoglycerate (1,3-BPG) is formed by the nucleophilic attack of Pi.on the carbonyl carbon.

14 Reaction 7, - the first ATP generation reaction

Phosphoglycerate kinase - the first ATP generation reaction

As in hexokinase, the 2 lobes of the enzyme close up upon substrate binding. This excludes water from the active site (why is this desirable for a phosphoryl transfer reaction?).

15 Coupling the GAPDH and PGK reactions

+ Reaction 6. GAP + Pi + NAD ↔ 1,3-BPG + NADH ∆G°’ = +6.7 kJ/mol

Reaction 7. 1,3-BPG + ADP ↔ 3PG + ATP ∆G°’ = -18.8 kJ/mol

Here the favorable PGK reaction seems like it “pulls” the slightly unfavorable GAPDH reaction. In the , ∆G for both reactions is approximately 0.

Reaction 8. Phosphoglycerate

Although on the surface this might look like a facile reaction, it is actually quite involved.

16 Proposed catalytic mechanism of A key aspect of this mechanism is the presence of a phospho- modified residue that is essential for .

Proposed catalytic mechanism of phosphoglycerate mutase If 2,3-BPG dissociated from the enzyme, would this enzyme now become inactive? How can this condition be reversed?

17 Reaction 9. Enolase-dependent generation of the second “high energy” product

This enzyme is divalent cation-dependent. Mg2+ is used most often, but other divalent cations will do as well. F- ions inhibit enolase by binding to magnesium. NaF inhibits glycolysis, and is therefore a potent poison.

Reaction 10. Generation of the second ATP and pyruvate by

1. Nucleophilic attack by the ADP β-phosphoryl oxygen on the PEP phosphorus generates ATP and enolpyruvate. ∆G°’ = -16 kJ/mol

2. Tautomerization of pyruvate to pyruvate. ∆G°’ = -46 kJ/mol, so this step drives the phosphoryl transfer reaction.

18 Schematic representation of the second stage of glycolysis

1. ATP. 2 ATP invested, 4 ATP returned.

2. NADH. 2 NAD+ are reduced into 2 NADH. NADH is used in oxidative phosphorylation to yield ATP.

3. Pyruvate. 2 pyruvate molecules are produced. Under aerobic conditions, pyruvate is used in the citric acid cycle reactions to yield additional ATP molecules. Under anaerobic conditions, pyruvate is used to regenerate NAD+.

Comparison of ∆G°’ values to cellular ∆G values

The ∆G°’ values are not particularly informative; rather, it is more useful to examine physiological reaction conditions.

In the , the ∆G values reveal that most reactions operate near equilibrium (∆G ≈ 0). Thus these reactions can be regulated by varying the concentration of reactants and products.

The hexokinase (1st), phosphofructokinase (3rd), and pyruvate kinase (10th) reactions operate far from equilibrium (∆G << 0). These reactions are essentially irreversible, thus set up a directional pathway. These reactions can be regulated by modulating the concentrations and activities of their respective enzymes, because these reactions are essentially 0-order with respect to substrates. Regulation of these enzymes can regulate the entire pathway.

19 Anaerobic fate of pyruvate Homolactic is used in the muscle of to convert pyruvate into lactate in a process that yields NAD+.

Under anaerobic glycolysis the overall set of reactions can be written:

Glucose + 2 ADP + 2 Pi → 2 lactate + + 2 ATP + 2 H2O + 2 H

Production of from pyruvate and bacteria do not have . Instead, they utilize pyruvate decarboxylase and dehydrogenase to regenerate NAD+ . This process, which yields ethanol, has been appreciated for quite some time. Pyruvate decarboxylase is not present in animal cells.

20 Thiamine pyrophosphate is an essential co-factor for pyruvate decarboxylase Thiamine pyrophosphate


Aminopyrimidine - ring H2 C C O P O P O C N H2 H2 N S O- O- Thiazolium ring H H3C N NH2 acidic

TPP (AKA thiamine diphosphate, ThDP) binds tightly, but non-covalently, to pyruvate decarboxylase. The thiazole ring acts as an electron sink in catalyzing the of pyruvate. In the absence of this electron “receiving” group, the transition state is not stable.

O O - O C O - O C O + C C R R

Proposed mechanism of pyruvate decarboxylase

1. Nucleophilic attack by TPP on the carbonyl carbon of pyruvate.

2. The thiazolium ring acts as an electron sink after the departure of CO2 to stabilize the intermediate.

3. protonation of the carbanion.

4. Release of and regeneration of the enzyme.

21 Thiamine pyrophosphate is an essential co-factor for many decarboxylases

Although pyruvate decarboxylase is not present in human cells, other decarboxylases are required for normal function. TPP is generated by phosphorylation of thiamine, which is B1. Thiamine is not stored in significant amounts in human cells and must be obtained on a regular basis form the .

Beriberi is an ultimately fatal condition that results from thiamine deficiency. The symptoms are neurological deficiencies presenting as pain, limb atrophy, and partial paralysis, leading to cardiac failure and/or lung edema and congestive heart failure.

Beriberi used to be common in Asia, where the thiamine-containing outer layers of rice often are removed before cooking, resulting in thiamine deficiency (now vitamin supplements can be taken to alleviate the disease). Beriberi sometimes is developed by alcoholics, who tend to drink but not eat regularly.